Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. The disclosure of these references are hereby incorporated by reference into the present disclosure, and for convenience are listed in the appended list of references.
Engagement of the T-cell antigen receptor (TCR) is directly coupled to the activation of non-receptor protein tyrosine kinases of both the Src and SYK/ZAP-70 families, leading to the phosphorylation of intracellular signaling proteins (1, 2). Among the downstream substrates of these activated kinases are signal-transducing enzymes such as phopholipase Cγ1 (PLCγ1), and adaptor proteins such as SLP-76 and LAT (linker of activated T-cells) (3, 4). Adaptor proteins play a critical role in mediating the formation of multi-protein signaling complexes and allowing propagation of the TCR signal (5). Phosphorylated LAT recruits SH2 domain containing protein PLCγ, Grb2 and Gads, while phosphorylated SLP-76 forms complexes with Vav, Nck and p130SLAP (ADAP) (4, 6, 7). Formation of these multi-protein complexes initiates a cascade of signaling events downstream of the TCR resulting in the up-regulation of IL-2 expression via activation of nuclear transcription factors such as nuclear factor of activated T-cells (NF-AT), reorganization of the actin cytoskeleton and adhesion (8). T-cell receptor activation thus leads to T-cell proliferation which is important in immune responses.
Adaptor proteins, that negatively regulate TCR signaling, are essential for the maintenance of T-cell homeostasis, the prevention of aberrant lymphocyte activation, and for regulating the duration of immune responses (2, 9). Adaptor proteins also function in assembling inhibitory complexes that play a role in mediating this down-regulation (10). Transmembrane proteins such as SIT and PAG, for example, recruit the tyrosine kinase Csk, to the membrane (1.1, 12). Csk acts as a negative regulator of the Src family kinases, Lck and Fyn, by phosphorylating the negative regulatory site found in the tail of these enzymes (13, 14). Cytosolic adaptors of the Dok family down-regulate activated antigen receptor complexes through recruitment of inhibitory molecules RasGAP, Csk and SHIP (15).
c-Cbl, is an ubiquitously expressed protein, initially characterized as an adaptor, that functions as a negative regulator of both receptor and non-receptor tyrosine kinases (16, 17). In addition to its adaptor function, c-Cbl also possesses a RING finger domain and has E3 ubiquitin ligase activity, which promotes ubiquitination of activated tyrosine kinases (16, 18, 19). Following TCR activation, c-Cbl is recruited to the activated TCR complex and tyrosine phosphorylated (20). C-activation of TCR signaling also leads to c-Cbl association with the SYK family kinases, SYK and ZAP-70 (21-23). The association between c-Cbl and the SYK family kinases results in a decrease in the activity and protein levels of these kinases (24-26), resulting in an overall down-regulation of signaling from the TCR. The mechanism by which c-Cbl negatively regulates SYK and ZAP-70 is not fully understood, however, it has been proposed that c-Cbl ubiquitin ligase activity may be involved in this process since the RING finger domain is essential for its inhibitory activity (27, 28).
Through its association with Zap-70, c-Cbl has been demonstrated to ubiquitinate the zeta chain of the TCR (29). Therefore, c-Cbl-mediated ubiquitination of components of the TCR could result in either degradation via the proteosome or alternatively, could serve as a signal for trafficking of the activated TCR complex to the lysosome. In agreement with this hypothesis, thymocytes from mice deficient in c-Cbl, exhibit both constitutively elevated tyrosine phosphorylation levels, and have increased cell surface TCR (25, 30). Additional support for this model derives from the observation that c-Cbl ubiquitinates and promotes the internalization and subsequent degradation of receptor protein tyrosine kinases (RPTKs) such as the epidermal growth factor receptor (EGFR) (31), the colony-stimulating factor-1 receptor (CSF-1R) (32), and the platelet-derived growth factor receptor (PDGFR) (33).
Another adaptor protein that negatively regulates signaling from the activated TCR is the Src-like adaptor protein (SLAP) (34). Originally identified as a protein that interacts with the cytoplasmic domain of EphA2, SLAP has been shown to be an inhibitor of mitogenic signals downstream of the PDGFR (35, 36). Subsequently, SLAP was found to inhibit both NFAT and AP-1 activation when transiently over expressed In Jurkat T cells (34). While the mechanism by which SLAP mediates inhibitory effects remains to be elucidated, it has been demonstrated that targeted disruption of the SLAP gene in mice results in increased surface TCR expression on double positive thymocytes (37).
The applicant has cloned and characterized a novel adaptor gene, MARS (Modulator of Antigen Receptor Signaling), which is a putative tumor suppressor gene and which exhibits structural and sequence similarity to SLAP.